Kit for analysis of subsets of subpopulations of leukocytes

ABSTRACT

A kit for determination of subsets of leukocytes utilizing flow cytometry analysis techniques.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of patent application Ser.No. 07/126,333, filed Nov. 30, 1987, now U.S. Pat. No. 4,987,086.

FIELD OF THE INVENTION

The present invention relates to a method and apparatus fordistinguishing and identifying subpopulations of cells in a bloodsample, and more particularly, relates to a method and apparatus fordistinguishing and quantifying subpopulations of lymphocytes in a samplethrough us of flow cytometry by gating on data retained from a priorsample.

DESCRIPTION OF THE PRIOR ART

Presently known and available flow cytometers useful for detectingparticles, cells and the like, commonly include one or more, usuallytwo, fluorescent channels for the detection of one or moresubpopulations of cells in a mixture. The flow cytometer also usuallyincludes means for determining cell volume, forward light scatter and/orside light scatter. The fluorescence channels are used to distinguishcells labeled with distinct fluorochromes which can be excited to emitlight at different wave lengths. Cell volume, forward light scatter andside light scatter measurements are used to separate classes of cellsbased upon physical parameters.

For example, flow cytometry devices are known which include twofluorescence detectors which can detect cells specifically labeled withtwo immunofluorescent agents associated with the respective fluorescencedetectors. In these known devices, a complete fluorescence detector,including the electrical circuitry and photo multiplier tube, is usedfor each type of fluorochrome labeled cells to be detected in themixture of cells in the sample being analyzed. Therefore, to detectmultiple subpopulations of cells in a sample using flow cytometry, anequivalent number of fluorescence detectors to fluorochromes is used.The cell volume, forward light scatter and side light scatter propertiesare also used to distinguish cells on the basis of physical parameters.Information can be obtained using a single pass of the cells through theflow cytometer and collecting data provided by the detection of separatefluorescence signals and physical parameters.

In the field of hematology in general, and in the specific field ofimmunohematology, it is desirable to determine the count of a variety ofwhite blood cells (leukocytes) which circulate in the peripheral blood.Subclassification of cells is performed and the count of cells in thesubpopulations is of great interest in the evaluation of immune relateddiseases, such as acquired immune deficiency syndrome (AIDS). Inparticular, the subpopulations of the lymphocytes, a mononuclear type ofleukocytes, has become of great clinical significance.

For example, it may be desirable to detect or quantify the totalpopulation of lymphocyte cells in the blood sample and to determine theproportion of T-cells and B-cells as a percentage of the lymphocytepopulation. Similarly, the detection and quantification of other typesof lymphocytes, such as the helper/inducer cell subset of T-cells andthe suppressor/cytotoxic subset of T-cells may be desired.

In performing an analysis on a sample of blood which includes all of theleukocytes, it is common for flow cytometry operators to estimate thebest gate that can be set which surrounds the population of lymphocytes.To establish this best gate by visual observation, a sample of the bloodcontaining the leukocytes is passed through the flow cytometer and theoperator observes the results on an oscilloscope using forward lightscatter (or cell volume) and side light scatter. By manipulating thecontrols of the flow cytometer (or data retention device) the operatorcan select (by drawing) a gate which will exclude cells which are notwithin the gate and will only retain information generated by cellswhich are within the gate. Typically, this is done by means of a"curser" whose movement is tied into the data retention device (i.e.,computer). The curser is moved to create the gate around the lymphocyteor other cell population of interest by tracing or circumscribing aline. Such establishment of a gate by visual observation is open tosubstantial error and substantial differences of observation betweendifferent operators.

With this in mind, the present invention is directed to satisfying adesired need for the direct determination of subpopulations of cells,and especially lymphocytes, from a blood sample, including substantiallyall of the leukocytes, by automatically establishing a cell populationgate without the need for manipulation of the flow cytometer by anoperator. It is, accordingly, the primary object of the presentinvention to provide a method of identifying and enumeratingsubpopulations of leukocytes while avoiding the necessity for theoperator to visually select a gate by observation of the passage ofcells in a sample through a flow cytometer. It is another object of theinvention to provide a method for analysis of leukocytes so as todistinguish and quantify populations of leukocytes which substantiallyprevents faulty analysis due to operator error. It is a further objectto provide a method for analysis of subpopulations of lymphocyteswherein the speed and relative simplicity involved renders lymphocytesubpopulation identification and enumeration a viable clinical tool.

SUMMARY OF THE INVENTION

The present invention is directed to a method and apparatus fordetermination of subsets of leukocytes utilizing flow cytometry analysistechniques. In accordance with one embodiment of the method foridentifying subpopulations of lymphocytes in a blood sample, a firstaliquot of a blood sample is provided which includes substantially allof the leukocytes. A first monoclonal antibody is labeled with a firstfluorochrome. The first monoclonal antibody reacts with substantiallyall the leukocytes in the sample. A second monoclonal antibody reactswith substantially all of the monocytes in the sample. The firstfluorochrome and the second fluorochrome have emission spectra which aredifferent.

A mixture of the first aliquot, the first labeled monoclonal antibodyand the second labeled monoclonal antibody then is prepared so as tocause the leukocytes to become labeled with the first monoclonalantibody and the monocytes to become labeled with the second monoclonalantibody. The first aliquot is then analyzed by flow cytometry so as todistinguish the lymphocyte population of the leukocytes. In thepreferred embodiment, data for each of the four parameters (i.e.,forward light scatter, side light scatter and fluorescence excitationenergy) is collected for each cell as it passes through the flowcytometer. The data for each cell in the first aliquot is stored in thedata retention device, and then is used to distinguish cell populations.Alternatively, cell volume may be used in place of forward lightscatter.

A second aliquot of the blood sample then is provided from which it isdesired to set a fluorescence markers and determine the percentage ofcells which are non-specifically stained. Fluorescence markers are theboundaries which delineate positive from negative fluorescence eventsfor each fluorochrome. The percentage of non-specific staining is thepercent of gated events from the first aliquot that fall in the positivefluorescence regions for this sample.

The second aliquot can be unlabeled or can be labeled with irrelevantmonoclonal antibodies which are not reactive with any leukocytes (e.g.,IgG, IgG₂ isotype monoclonals) to establish the fluorescence markers.Preferably, the second aliquot is labeled and is then analyzed by flowcytometry using forward light scatter, side light scatter andfluorescentexcitation energy. The gate from the first aliquot then isapplied. Thus, only cells having scatter characteristics within thelymphocyte gate are analyzed in the second aliquot. Because irrelevantmonoclonal antibodies are used, none of the cells in the lymphocyte gateshould be specifically labeled. Accordingly, the fluorescence markerspositions are set to define "negative" and "positive" boundaries forboth FL1 and FL2 and also define the percentage of cellsnon-specifically stained. The information obtained from the analysis ofthe second aliquot is retained, as above.

A third aliquot of the blood sample then is provided from which it isdesired to analyze any of the various subpopulations of cells,preferably lymphocytes. A third monoclonal antibody which is reactivewith a first subpopulation of lymphocytes is labeled with a thirdfluorochrome. A fourth monoclonal antibody which is specific for asecond subpopulation of lymphocytes is labeled with a fourthfluorochrome. It is understood that the third fluorochrome and thefourth fluorochrome can be identical to the first and secondfluorochromes previously described for use in analyzing the firstaliquot of the sample and that other subpopulations of lymphocytes alsoma be labeled by the third and fourth monoclonal antibodies.

A mixture of the third aliquot, the third labeled monoclonal antibodyand the fourth labeled monoclonal antibody is then prepared so as tocause the third monoclonal antibody to label a first lymphocytesubpopulation and the fourth monoclonal antibody to label a secondlymphocyte subpopulation and the fourth monoclonal antibody to label asecond lymphocyte subpopulation.

Preferably, the third sample aliquot then is analyzed by flow cytometryusing forward light scatter, side light scatter and fluorescentexcitation energy so as to distinguish the subpopulations of lymphocytesrecognized by the third monoclonal antibody and the fourth monoclonalantibody. The third aliquot analysis is performed on the basis of thelymphocyte gate and fluorescence markers established by the first andsecond sample aliquot analysis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 comprises several dot plots of SSC versus FSC (A), FL2 versus FL1(B), FL2 versus SSC (C) and FL1 versus FSC (D) for a sample of cellstaken from erythrocyte lysed whole blood labeled with Anti-HLe 1 (FITC)and Anti-Leu M3 (PE) wherein X represents the SSC channel thatdelineates the boundary between lymphocytes (including debris) and othercells, Y represents the FSC channel that delineates the boundary betweencells and debris, X' represents the FL2 channel that delineates theboundary between cells (including debris) and monocytes and Y'represents the FL1 channel that delineates the boundary betweenlymphocytes and cells (including debris), wherein the selection of X andY are set so as to include the maximum number of lymphocytes whileminimizing contamination from other cells or debris, and further whereinthe figure comprises a dot plot of FL2 versus FL1 (E) of the cellsfalling within the boundaries set in (A).

FIG. 2 is a dot plot of FL2 versus FL1 (A) and two histograms (one foreach parameter, B-C respectively) for cells in the second aliquot whichhave been stained with irrelevant isotype control monoclonal antibodieslabeled with PE and FITC and which fall within the boundaries set forthe lymphocyte gate in FIG. 1 wherein X" and Y" represent an upperboundary for FL2 and FL1 respectively that includes greater than 95% ofall cells.

FIG. 3 comprises two dot plots of cells in the third aliquot which havebeen labeled with Anti-Leu 4 (FITC) and Anti-Leu 12 (PE) monoclonalantibodies wherein cells (as above) are plotted for SSC versus FSC (A)and only the cells falling within the lymphocyte gate from FIG. 1(A) arereplotted for FL2 versus FL1 (B) and wherein the fluorescence markersfrom FIG. 2(b) are used to define positive and negative fluorescenceboundaries.

FIG. 4 and FIG. 5 represent flow cytometry results obtained fromanalysis of a first aliquot of a blood sample.

FIG. 6 and FIG. 7 represent flow cytometry results obtained fromanalysis of a second aliquot of the same sample.

FIG. 8 and FIG. 9 represent flow cytometry analysis of a third aliquotof the same sample.

The scope of the present invention will become more apparent from thefollowing detailed description and the accompanying claims.

DETAILED DESCRIPTION

While the present invention is satisfied by embodiments in manydifferent forms, there is shown in the drawings and will herein bedescribed in detail a particular embodiment of the invention, with theunderstanding that the following description is to be considered asexemplary of the principles of the invention and is not intended tolimit the scope of the invention as determined by the appended claimsand their equivalent.

The present invention provides a method for identifying and enumeratingcells in subpopulations of leukocytes in a population of interest in ablood sample utilizing a gate established in a flow cytometry apparatusby analysis of the entire blood sample. This gate excludes observationof cells which are not within the population of interest. Forconvenience of discussion, the blood sample described is lysed wholeblood which contains substantially all leukocytes and the population ofinterest is the lymphocytes.

In the method as depicted in FIGS. 1-3, a first aliquot of a bloodsample is provided which includes substantially all of the leukocytes.Referring to FIG. 1, a first monoclonal antibody is labeled with a firstfluorochrome, which is fluorescein isothiocyanate (FITC). The firstmonoclonal antibody, which is Anti-HLe-1, reacts with substantially allthe leukocytes in the sample. Anti-HLe-1 and all monoclonalantibodiesreferred to herein, unless otherwise noted, are commercially availablefrom Becton Dickinson Immunocytometry Systems, San Jose, California. Asecond monoclonal antibody is labeled with a second fluorochrome, whichis phycoerythrin (PE). The second monoclonal antibody, which isAnti-Leu-M3, reacts with substantially all of the monocytes in thealiquot. The first fluorochrome and the second fluorochrome haveemission spectra which are different.

A mixture of the first aliquot, the first labeled monoclonal antibodyand the second labeled monoclonal antibody is then prepared so as tocause the leukocytes to become labeled with the first monoclonalantibody and the monocytes to also become labeled with the secondmonoclonal antibody. It will be appreciated from FIG. 1 that eachpopulation of cell will react differentially with each of the antibodiessuch that the level of fluorescence emissions can be used to distinguishbetween cells. The first aliquot is then analyzed by flow cytometry witha FACScan™ flow cytometer (Becton Dickinson Immunocytometry Systems)using forward light scatter, side light scatter and fluorescentexcitation energy so as to distinguish the lymphocyte population of theleukocytes and provide a lymphocyte gate. The parameters X and Y areselected so as to delineate the boundary between lymphocytes and othercell populations (including debris). See FIGS. 1 (C,D). Selection of thespecific channel by the algorithm includes the use of X' and Y' whichfurther distinguishes one population from another resulting in a gatethat maximizes the number of lymphocytes within the gate whileminimizing contamination. The information obtained from the analysis ofthe first aliquotis retained, preferably in a computer (e.g., a Consort30 Data Management System, Becton Dickinson Immunocytometry Systems).The analysis of the first aliquot also provides information concerningthe percentage of monocytes, granulocytes and debris within thelymphocyte gate. See FIG. 1(B).

In the preferred embodiment of the invention, a second aliquot of theblood sample is analyzed by flow cytometry to determine the fluorescencemarkers for the sample. See FIG. 2. To determine the fluorescencemarkers, the second aliquot is passed through the flow cytometer anddata collected for the forward light scatter, side light scatter andfluorescent excitation parameters. The data are analyzed by applying thelymphocyte gate from the first aliquot so that only information from thecells falling within the boundaries of SSC and FSC set by the lymphocytegate is used to determine the fluorescence markers. The boundaries forthe markers again can be set by an algorithm which is calculated toinclude 95% of the cells on the fluorescence histograms. See FIGS. 2(B,C). The use of this second aliquot is preferred since data relativeto background fluorescence (i.e., non-specific staining) can be moreprecisely quantified. The fluorescence marker information can be furtheroptimized by use of two labeled irrelevant monoclonal antibodies whichdo not react with any leukocytes, one of which is IgG₁ monoclonalantibody labeled with FITC and the other of which is an IgG₂ monoclonalantibody labeled with PE.

If a second aliquot is analyzed as in the preferred embodiment, a thirdaliquot of the blood sample then is provided from which it is desired toanalyze any of various subpopulations of lymphocytes. If, however, thesecond aliquot is omitted, the "third" aliquot is used to establish thefluorescence markers as well as to analyze the various subpopulations oflymphocytes and may be defined as the second aliquot.

In a specific example of the preferred embodiment, a third monoclonalantibody which reacts with a first subpopulation of lymphocytes, such asAnti-Leu-4 (i.e., anti-CD3) which reacts with all T-cells, is labeledwith FITC, as shown in FIG. 1. A fourth monoclonal antibody which reactswith a second subpopulation of lymphocytes, such as Anti-Leu 12 (i.e.,anti-CD19) which is specific for B-cells, is labeled with anotherfluorochrome, such as PE, as shown in FIG. 3.

A mixture of the third aliquot, the third labeled monoclonal antibodyand the fourth labeled monoclonal antibody then is prepared so as tocause the third monoclonal antibody to label a first lymphocytesubpopulation (T-cells) and the fourth monoclonal antibody to label asecond lymphocyte subpopulation (B-cells). The third aliquot then isanalyzed by flow cytometry using forward light scatter, side lightscatter and fluorescence excitation energy so as to distinguish thesubpopulations of lymphocytes recognized by the third monoclonalantibody and the fourth monoclonal antibody. The third aliquot analysisis performed on the basis of alymphocyte gate established by the firstaliquot analysis and the fluorescence markers established by the secondaliquot analysis.

In the context of the present invention, monoclonal antibodies are usedas marking agents. A tagging agent comprises a light emitting agentwhich alone or in combination with a marking agent can label astructure(s) in or on a cell. The tagging agents for marking the variouscell populations in the method of the invention are preferablymonoclonal antibodies conjugated to a suitable fluorochrome. Othersuitable tagging agents are lectins, nucleic acid dyes, such aspropidium iodide, and membrane potential dyes, such as cyanine dyes.Some tagging agents, such as monoclonal antibodies, react with antigenicsites on the surface of the cells. Other tagging agents, such as nucleicacid dyes and membrane potential dyes are absorbed by a specificcellular component so as to mark the subpopulation or population ofcells of interest.

Fluorescence emission of certain tagging agents which do notspontaneously emit detectable light (such as monoclonal antibodies) maybe obtained by coupling a marking agent to a suitable emitting agent,such as a fluorochome. Some tagging agents (such as nucleic acid dyesand membrane potential dyes) have fluorescent properties andspontaneously emit detectable light upon optical stimulation. For suchcases, a coupled marking agent is not required. The emitting agents foruse with non-spontaneously emitting marking agents are preferably two ormore fluorochromes which have emission spectra with sufficient wavelength separation to permit simultaneous detection of two or morecolors. In general, the fluorochromes should emit fluorescence in therange of from about 10 to about 300 nm above the excitation wave length.One particularly suitable pair of marking agents has been found to bephycoerythrin, (PE), a phycobiliprotein which emits fluorescence at 570nm and fluorescein isothiocyanate (FITC) which emits fluorescence at awave length of 530 nm. Both PE and FITC are excited at the 488 nmwavelength. Three or more suitable tagging agents can be used foranalysis of aliquots if they have suitably distinct emission propertiesor are tagged with emitting agents having suitably distinct emissionproperties. One suitable group of three fluorochromes for coupling toantibodies are phycoerythrin, fluorescein isothiocyanate, and aphycoerythrin-texas red conjugate which emits fluorescence at 615 nm.One or more of these marking agents may be used in combination with anucleic acid dye, such as propidium iodide.

If whole blood is used, the incubated aliquot is passed through a flowcytometry apparatus at a white blood cell flow rate that issubstantially equivalent to the white blood cell flow rate that isnormally used for samples wherein the red blood cells are removed orlysed. In general, flow rates of from about 20 to about 1000 white bloodcells/sec. are suitable. Preferably, the aliquot is passed through theflow cytometry apparatus following lysis or separation of the whiteblood cells from the red blood cells by method known to those skilled inthe art (e.g., FACS™ Lysing Solution, Becton Dickinson ImmunocytometrySystems). It should be understood, however, that the principles of thepresent invention are equally suitable for the analysis of blood cellfractions which include the white blood cells which are provided bylysis of red blood cells or separation of specific populations of bloodcells from the sample under investigation, as well as from whole bloodcell preparations including red blood cells and white blood cells.

The subpopulations of lymphocytes which can be distinguished by themethod of the invention include T-cells, helper T-cells, inducerT-cells, suppressor T-cells, cytotoxic T-cells, T-cells inmaturation/activation stages, B-cells, B-cells in maturation/activationstages, NK/LGL cells and NK/LGL cells in maturation/activation stages.Of course, the method of this invention can be used to distinguish andcompare two or more sets of the subpopulations enumerated hereinabove.

By suitable selection of the second monoclonal antibody and secondfluorochrome, populations of cells other than lymphocytes, such asgranulocytes and monocytes, can be examined. Like the lymphocytepopulation, within each of the other populations, other subpopulationscan be examined. For example, if the population is granulocytes, thesubpopulations can be of any subclass of T-cells or a combinationthereof; if the set is granulocytes, the subclass can be neutrophils,eosinophils, basophils, maturation stage granulocytes and combinationsthereof.

In particular, a preferred population of cells is lymphocytes and thefollowing pairs of lymphocyte subpopulations:

(1) T-cells and B-cells;

(2) CD4 bearing cells and cells expressing the Leu 8 antigen;

(3) T-cells and activation antigens expressed by T-cells;

(4) CD4 bearing cells and CD8 bearing cells; and

(5) CD8 bearing cells and cells expressing the Leu 7 antigen (i.e.,CD57).

The "CD" designation for antigens is the International standard forleukocyte antigens for which monoclonal antibodies have been developed.The "Leu" designation for antigens is the Becton DickinsonImmunocytometry Systems system for antigens for which monoclonalantibodies have been developed and commercialized.

It is understood that the present invention is useful in many types offlow cytometry devices which measure light scatter, particle or cellvolume, fluorescence or any other optical parameters for theidentification or quantification of subpopulations of particles in asample. For example, the elements of a suitable flow cytometry apparatusare included in a FACScan™ flow cytometer. The FACScan™ flow cytometeranalyzes cell populations on the basis of light scatter and fluorescencein a wide variety of research and laboratory applications. Other detailsof a cell analysis and sorting apparatus useful in conjunction with thepresent invention are described in U.S. Pat. No. 3,826,364.

In accordance with one embodiment of the invention, a computer (e.g.,Consort 30) is provided to retain the results of the analysis of thefirst and second aliquots of the blood sample to provide the lymphocytegate and fluorescence markers which are used in the analysis of thesubsequent aliquots of the blood sample. When two labeled antibodies areused, a two dimensional plot of fluorescence properties of the cellpopulations is generated. When three labeled antibodies are used arepresentational three dimensional plot can be generated or a series oftwo dimensional plots can be generated showing subpopulations of cellsin groups of two.

The present invention is also directed to providing a kit for use in thepractice of the method of the invention. In its simplest form the kitincludes a vial or other suitable container filled with a mixture of afirst monoclonal antibody coupled to a first fluorochrome and a secondmonoclonal antibody coupled to a second fluorochrome. The firstmonoclonal antibody is specific for substantially all leukocytes and thesecond monoclonal antibody is specific for substantially all monocytes.The first and second fluorochromes have different light emissionproperties, as described hereinabove.

The kit can also contain one or more additional vials containing one ormixtures of one or more monoclonal antibodies coupled to suitablefluorochromes. In each additional vial, there is most commonly a set oftwo monoclonal antibodies reactive with particular subpopulations oflymphocytes. The following examples further illustrate various featuresof the invention but are intended to in no way limit the scope of theinvention for detecting, distinguishing and/or quantifyingsubpopulations of lymphocytes.

EXAMPLE 1

In this example, as exemplified in FIGS. 4-9, two components or subsetsof lymphocytes, (i.e., T-cells and B-cells) were determined as apercentage of total lymphocytes from a human blood source.

For the first analysis an aliquot of 0.05 cc (50 microliters) ofsuspended peripheral blood cells were added to 0.02 cc of a mixture of afirst monoclonal antibody Anti-HLe-1 (i.e., anti-CD45) conjugated tofluorescein isothiocyanate, and a second monoclonal antibody Anti-Leu-M3(i.e., anti-CD14) conjugated to phycoerythrin. The aliquot wasmaintained at room temperature at the time of addition and thereafterfor one-half hour. After one-half hour of incubation, a further additionof 3 ml of a red cell lysing solution (e.g., FACS™ Lysing Solution) wasmade and the sample was mixed and held for 10 minutes.

The first aliquot was passed through a flow cytometry apparatus (e.g.,FACScan™), and was analyzed to provide forward light scatter, side lightscatter, fluorescence one channel data and fluorescence two channeldata. A representation of the results obtained from analysis of thefirst aliquot is shown in FIG. 4 and FIG. 5. The data generated byanalysis of the first aliquot was retained in a computer (e.g., Consort30) having an algorithm (as described above) designed to analyze thedata using all four parameters and provide a lymphocyte gate around thegroup of lymphocyte cells.

As shown in FIG. 4, the results of this first analysis using forwardlight scatter and side light scatter are isolation of four populationsof cells. One population represents monocytes, one population representsgranulocytes, one population represents debris and the other populationare the cells of interest (i.e., the lymphocytes). As shown in FIG. 5,the analysis of the two colors, phycoerythrin and fluoresceinisothiocyanate, also provide four populations of cells. As shown in FIG.1, the four populations are the same as the four populations generatedby analysis of forward light scatter and side light scatter. Thealgorithm used with the computer is designed to compare the datagenerated by light scatter, as shown in FIG. 4 and the data generated byfluorescence analysis, as shown in FIG. 5 and to differentiate thosecells which should be included within the lymphocyte gate. The objectiveis to set a gate around the lymphocyte population using the physicalparameters, forward and side light scatter. The physical properties(FIG. 4) and the fluorescence (FIG. 5) properties are used to determinethe location of the lymphocytes on the forward light scatter vs. sidelight scatter plot. Any interference from debris, monocytes orgranulocytes that exist is quantified and reported but cannot removedfrom consideration. Since the physical properties for the sample willremain constant for all aliquots of the sample, the lymphocyte gate isapplied and subsequent analysis of a second or further aliquots of thesample need only contain the fluorescent plot for the monoclonalantibodies used for the lymphocyte subsets of interest.

As shown in FIG. 2 and using the preferred embodiment, a second aliquotcontaining isotype control monoclonal antibody reagents is prepared. TheIgG₁ monoclonal antibody is conjugated to fluorescein isothiocyanate andthe IgG₂ monoclonal antibody is conjugated to phycoerythrin.

FIGS. 6 and 7 depict the four parameter representation of the secondaliquot after incubation with the IgG₁, and IgG₂ antibodies and passagethrough a flow cytometry apparatus. FIG. 6 shows the lymphocyte gate setby the data retained from the analysis of the first aliquot and FIG. 7shows the fluorescence markers that are calculated using the gatedfluorescence data for this sample. Non-specific staining is the percentof gated events which fall in the upper left, upper right, and lowerright quadrants. The quadrant counts are corrected for interference frommonocytes, granulocytes or debris that was quantified in the firstsample aliquot.

As shown in FIG. 3, a third aliquot containing T-cell and B-cellmonoclonal antibody reagents is prepared. The T-cell monoclonal antibodyreagent is Anti-Leu 4 conjugated to fluorescein isothiocyanate and theB-cell monoclonal antibody reagent is Anti-Leu-12 conjugated tophycoerythrin.

FIGS. 8 and 9 depict the four parameter representation of the thirdaliquot after incubation with the Leu-12 and Leu-4 monoclonal antibodiesand passage through a flow cytometry apparatus. FIG. 8 shows thelymphocyte gate set by the data retained from the analysis of the firstaliquot and FIG. 9 shows the fluorescence markers set by the secondaliquot analysis and gated fluorescence data for the third aliquot.T-cells are in the lower right quadrant of FIG. 9, B-cells are in theupper left quadrant and T⁺ /B⁺ cells are in the upper right quadrant.The quadrant counts are corrected for interference from monocytes,granulocytes or debris that was quantified in the first sample aliquot.

EXAMPLE 2

A blood sample was again analyzed for T⁺ /B⁺ as a percentage of totallymphocytes in accordance with the procedure of Example 1.

In this example, the second aliquot analysis was omitted. Thefluorescence markers were calculated from the third aliquot whereinAnti-Leu-12 and Anti-Leu-4 were used. Other suitable combinations ofmonoclonal antibodies useful in establishing fluorescence markersinclude Anti-Leu-3 and Anti-Leu-2.

The present invention provides a method for analysis of subpopulationsof lymphocytes without risk of operator error through establishment ofan improper lymphocyte gate for the flow cytometry apparatus.

What is claimed:
 1. A kit for determination of subsets of asubpopulation of leukocytes in a sample containing a first subpopulationof leukocytes of interest and a second subpopulation of leukocytes whichare not of interest, said kit comprising; a first monoclonal antibodywhich is labeled with a first fluorochrome and which reacts withsubstantially all leukocytes, a second monoclonal antibody which islabeled with a second fluorochrome and reacts with at least a subset ofleukocytes, a third monoclonal antibody which is labeled with a thirdfluorochrome and which reacts with a first subset of leukocytes withinsaid first subpopulation of interest and a fourth monoclonal antibodywhich is labeled with a fourth fluorochrome and which reacts with asecond subset of leukocytes within said first subpopulation of interest,said first and second fluorochromes and said third and fourthfluourochromes having distinct and different emission spectra.
 2. A kitin accordance with claim 1 which also includes an IgG₁ monoclonalantibody and an IgG₂ monoclonal antibody.
 3. A kit in accordance withclaim 1 or 2 wherein said second monoclonal antibody reacts withsubstantially all monocytes.
 4. A kit in accordance with claim 1 or 2wherein said third monoclonal antibody reacts with substantially allT-cells and said fourth monoclonal antibodies reacts with substantiallyall B-cells.
 5. A kit in accordance with claim 1 or 2 wherein said thirdmonoclonal antibody reacts with substantially all T-cells and saidfourth monoclonal antibody reacts with at least one activation antigenexpressed by T-Cells.
 6. A kit in accordance with claim 1 or 2 whereinsaid third monoclonal antibody reacts with substantially all CD4 bearingcells and said fourth monoclonal antibody reacts with substantially allcells expressing the Leu 8 antigen.
 7. A kit in accordance with claim 1or 2 wherein said third monoclonal antibody reacts with substantiallyall CD4 bearing cells and said fourth monoclonal antibody reacts withsubstantially all CD8 bearing cells.
 8. A kit in accordance with claim 1or 2 wherein said third monoclonal antibody reacts with substantiallyall CD8 bearing cells and said fourth monoclonal antibody reacts withsubstantially all cells expressing the Leu 7 antigen.